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Use of in vitro assays to identify antibiotics that are cytotoxic to normal equine chondrocytes and synovial cells.

使用体外测定法鉴定对正常马软骨细胞和滑膜细胞具有细胞毒性的抗生素。

  • 影响因子:0
  • DOI:10.1111/evj.13314
  • 作者列表:"Pezzanite L","Chow L","Piquini G","Griffenhagen G","Ramirez D","Dow S","Goodrich L
  • 发表时间:2021-05-01
Abstract

BACKGROUND:Intra-articular (IA) antibiotic usage is prevalent in equine practice. However, recent emergence of antimicrobial resistance prompts re-evaluation of antibiotic selection, particularly when used prophylactically. Furthermore, many commonly used antibiotics exert direct cytotoxicity to equine cells, and appropriate IA doses have not been defined. OBJECTIVES:To screen antibiotics in vitro as an initial assessment of cytotoxicity against normal equine joint cells in monolayer culture and explant tissues. STUDY DESIGN:In vitro experimental study. METHODS:Chondrocytes and synovial cells were harvested from three horses and plated on 24-well plates (100 000 cells/wells in triplicate) for 48 hours prior to addition of antibiotics. Joint cells were exposed to antibiotics (n = 15) at various doses (25-0.39 mg/mL in complete DMEM media) for 24 hours and viability was assessed by trypan blue dye exclusion. The half maximal inhibitory concentration (IC50) was determined for each antibiotic. Cartilage explants were obtained from 3 horses, minced and exposed to antibiotics (n = 5) for 72 hours. Live/dead staining was performed, and fluorescence was visualised using Olympus IX83 spinning disk confocal microscope. Percentage of live vs dead cells was quantified. RESULTS:Antibiotics from different antimicrobial classes expressed dose-dependent but variable cytotoxicity to equine joint cells in vitro. Aminoglycosides and doxycycline had the lowest IC50 (most toxic). Ampicillin sulbactam, imipenem, tobramycin, ceftiofur sodium and amoxicillin had IC50 > 25 mg/mL for at least one cell line, representing potentially less cytotoxic alternatives. MAIN LIMITATIONS:Further studies are necessary to extrapolate these in vitro data results to the in vivo joint environment. CONCLUSIONS:Targeted IA antibiotic therapy would involve selection of the safest antibiotics (highest IC50) with efficacy based on bacterial culture/sensitivity. Antimicrobial selection and evidence-based dosing may minimise damage to native articular cartilage and synovial cells and development of antimicrobial resistance when IA antibiotics are used in equine practice.

摘要

背景: 在马的实践中,关节内 (IA) 抗生素的使用是普遍的。然而,最近出现的抗微生物剂耐药性促使重新评估抗生素选择,特别是当预防性使用时。此外,许多常用的抗生素对马细胞具有直接的细胞毒性,并且还没有确定合适的IA剂量。 目的: 体外筛选抗生素作为对单层培养和外植体组织中正常马关节细胞的细胞毒性的初步评估。 研究设计: 体外实验研究。 方法: 从三匹马收获软骨细胞和滑膜细胞,并在添加抗生素之前在24孔板 (100细胞/孔,一式三份) 上铺板48小时。将关节细胞暴露于各种剂量 (25-0.39 mg/ml,在完全DMEM培养基中) 的抗生素 (n = 15) 24小时,并通过锥虫蓝染料排除法评估活力。测定每种抗生素的半数最大抑制浓度 (IC50)。从3匹马获得软骨外植体,切碎并暴露于抗生素 (n = 5) 72小时。进行活/死染色,并使用Olympus IX83旋转盘共聚焦显微镜使荧光可视化。对活细胞与死细胞的百分比进行定量。 结果: 来自不同抗菌类别的抗生素在体外对马关节细胞表现出剂量依赖性但可变的细胞毒性。氨基糖苷类和多西环素的IC50最低 (毒性最强)。氨苄青霉素舒巴坦、亚胺培南、妥布霉素、头孢噻呋钠和阿莫西林对至少一种细胞系具有> 25 mg/mL的IC50,代表潜在的较少细胞毒性的替代物。 主要限制: 需要进一步研究以将这些体外数据结果外推至体内关节环境。 结论: 靶向IA抗生素治疗将涉及基于细菌培养/敏感性选择具有功效的最安全抗生素 (最高IC50)。当在马实践中使用IA抗生素时,抗微生物剂选择和基于证据的给药可以最小化对天然关节软骨和滑膜细胞的损伤以及抗微生物剂耐药性的发展。

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